(T1330-10-59) Statins Constrict Resistance-Size Cerebral Arteries by Stimulating L-type Ca²⁺ Channels

Purpose: Statins are amongst the most widely prescribed drugs in the world with a range of vascular effects that have been primarily attributed to the inhibition of cholesterol and mevalonate biosynthesis, and the inhibition of mevalonate-dependent Rho/ROCK signaling upon long-term treatment. However, no studies have investigated the direct effects of acute statin application on fresh isolated resistance cerebral arteries using therapeutic concentrations of statins. Here, we examined acute vascular effects of therapeutically relevant concentrations (0.01-10nM) of rosuvastatin and simvastatin on Sprague Dawley rat cerebral arteries and underlying molecular mechanisms.

Methods: We used pressurized arterial myography, simultaneous of vessel Ca²⁺ fluorescence and diameter measurement, pharmacological modulators and RNAi approaches. Cannulated arterial segments were maintained at 37°C in a perfusion chamber and intraluminal pressure slowly increased to 60 mmHg. Simvastatin, rosuvastatin (0.01-10nM) and pharmacological modulators were applied after the development of myogenic tone at 60 mmHg and diameter changes tracked. For calcium imaging, the isolated arteries were preincubated in Fura-2AM (10μM) and 0.02% Kolliphor solution at room temperature for two hours before mounting in the perfusion chamber. siRNA knockdown of CaV1.2 was performed by electroporation and confirmed by Western blotting.

Results: At 60mmHg, cerebral arteries developed ~36% myogenic tone, after which increasing concentrations of statins were applied. Our data showed that the application of 1nM rosuvastatin and simvastatin constricted cerebral arteries by ~26 μm and ~24μm, respectively, within 2-3 minutes of drug application. Such statin-induced vasoconstriction remained unaltered upon endothelium denudation (intact ~23μm vs denuded ~25μm), suggesting an endothelium-independent mechanism. Co-application of mevalonate did not alter the vasoconstriction either (control ~28μm vs mevalonate ~29 μm), indicating that the effect is HMG-CoA reductase-independent. However, removal of extracellular Ca²⁺ with EGTA or the application of nimodipine a selective blocker of smooth muscle cell voltage-gated Ca²⁺ channel, Ca_V1.2, each abolished cerebral artery vasoconstriction by statins, indicating that the Ca²⁺ entry through Ca_V1.2 plays a critical role here. We found that co-application of ryanodine, a blocker of ryanodine receptor-mediated Ca²⁺ release, had no effects on statin-induced constriction (control ~27μm vs ryanodine ~26 μm). In contrast, statin-evoked vasoconstriction of cerebral arteries was significantly attenuated upon co-application of thapsigargin, a blocker of SR/ER membrane Ca²⁺-ATPase pump (control ~27μm vs thapsigargin ~18 μm). siRNA knockdown of Ca_V1.2 channels reduced protein abundance and abolished statin-evoked constriction in cerebral arteries. Simultaneous measurement of arterial Ca²⁺ fluorescence and diameter further confirmed the involvement of Ca_V1.2 channel in mediating Ca²⁺ entry and subsequent Ca²⁺ release, leading to cerebral artery vasoconstriction.

Conclusion: Altogether, our data suggests that smooth muscle cell Ca_V1.2 opening and Ca²⁺ influx is the primary mechanism underlying statin-induced constriction in cerebral arteries. Vasoconstriction does not depend on HMG-CoA reductase and mevalonate-dependent pathways suggesting that this effect is directly mediated by an unconventional molecular target.

References: 1. Liao et al., Pleiotropic Effects of Statins. Annu Rev Pharmacol Toxicol 45:89-118 (2005)
2. Oesterle A et al., Pleiotropic Effects of Statins on the Cardiovascular System. Circ Res 120:229-243 (2017)
3. Rohilla A et al., Pleiotropic Effects of Statins: A boulevard to cardioprotection. Arab J Chem 9:S21S25 (2016)
4. Hasan R, Jaggar JH. KV channel trafficking and control of vascular tone. Microcirc 25:e12418 (2018)
5. Bulley S, Fernandez-Pena C, Hasan R et al., Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure. eLife 7:e42628 (2018)
6. Absi M, Eid BG, Ashton N, Hart G, Gurney AM. Simvastatin causes pulmonary artery relaxation by blocking smooth muscle ROCK and calcium channels: Evidence for an endothelium-independent mechanism. PloS one. 2019;14(8)

Acknowledgements: This work was funded by a NIH/NHLBI research grant 1R15HL156138- 01A1 and a start-up grant from the Mercer University College of Pharmacy to Raquibul Hasan, PhD.